Hyaluronic acid, also referred to as xe2x80x9cHA,xe2x80x9d is a naturally occurring, water soluble polysaccharide comprising disaccharide units of D-glucuronic acid (GlcUA) and N-acetyl-D-glucosamine (GlcNAc), which are alternately linked, forming a linear polymer. High molecular weight HA may comprise 100 to 10,000 disaccharide units. HA often occurs naturally as the sodium salt, sodium hyaluronate. HA, sodium hyaluronate, and preparations of either HA or sodium hyaluronate are often referred to as xe2x80x9chyaluronan.xe2x80x9d As used herein, the terms xe2x80x9cHAxe2x80x9d and xe2x80x9chyaluronanxe2x80x9d also refer to any of the other hyaluronate salts, including, but not limited to, potassium hyaluronate, magnesium hyaluronate, and calcium hyaluronate.
HA is a major component of the extra-cellular matrix and is widely distributed in animal tissues. Naturally occurring HA generally has a molecular weight range of about between 6xc3x97104 to about 1.2xc3x97107 daltons. It has excellent biocompatibility and does not give a foreign body reaction when implanted or injected into a living body. An aqueous solution of hyaluronan is viscous even at relatively low solute concentrations.
Methods of preparing commercially available hyaluronan are well known. Also known are various methods of coupling HA and cross-linking HA to reduce the water solubility and diffusibility of HA, and to increase the viscosity of HA. See, for example, U.S. Pat. Nos. 5,356,883 and 6,013,679, the teachings of which are incorporated herein by reference in their entireties.
Chemically modified HA has been used as a surgical aid to prevent post-operative adhesions of tissues.
Currently there is interest in developing chemically modified HA for delivery of bioactive agents including, for example, therapeutic agents or drugs and biological probes. A major challenge is the development of a delivery vehicle that will provide the appropriate level of bioavailability of a therapeutic agent at the affected area to achieve a desired clinical result. The bioavailability of a drug depends upon the nature of the drug, the drug delivery vehicle used, and the route of delivery, for example, oral, topical, transdermal, mucosal, administration by injection, administration by inhalation, or administration by a combination of two or more of these routes. The bioavailability may be low as a result of, for example, the degradation of the drug by stomach acid, elimination from the gastrointestinal tract, or high aqueous solubility of the drug. As a result, frequent administration may be required, and the amount of drug delivered with each administration may be high, leading to an increase in the occurrence of damaging side effects.
Highly viscous cross-linked HA derivatives are sometimes used as an aid in ophthalmic surgery, such as intraocular lens implantation, glaucoma surgery, vitrectomy, and repair of retinal detachment. However, because of its high viscosity and stability, this cross-linked HA does not readily clear out through the trabecular meshwork, the outlet for aqueous humor egress. Blockage of the trabecular meshwork by the cross-linked HA may contribute to post-operative increases in intraocular pressure, including intraocular spikes (IOPs), the increases in pressure sometimes causing damage to the optic nerve, as well as damage to the cornea.
Cross-linked HA that is highly viscous is also used as a scaffold for tissue engineering in vitro or guided tissue regeneration or augmentation in vivo. Because of the high viscosity and stability of this HA derivative, however, recovery of cells grown on the cross-linked HA can be problematic.
The present invention relates to compositions including, for example, a biscarbodiimide having an intramolecular disulfide bond. The invention inter alia also includes the following embodiments, alone or in combination. In one embodiment, a biscarbodiimide having an intramolecular disulfide bond is formed by a method including reacting an isothiocyanate with cystamine (2,2xe2x80x2-dithiobis(ethylamine), (H2NCH2CH2)2S2), thereby forming a thiourea derivative, which is then reacted with an oxidizing agent or a dehydrosulfuration agent, thereby forming a biscarbodiimide having an intramolecular disulfide bond.
In another embodiment, a biscarbodiimide having an intramolecular disulfide bond is formed by a method including reacting an isocyanate with cystamine, thereby forming a urea derivative, which is then reacted with a dehydrating agent, thereby forming a biscarbodiimide having an intramolecular disulfide bond.
In a particular embodiment, the biscarbodiimide having an intramolecular disulfide bond is represented by Structural Formula (1): 
In another embodiment, a biscarbodiimide having an intramolecular disulfide bond is formed by a method including reacting an isothiocyanate with 2-aminophenyl disulfide or 4-aminophenyl disulfide, thereby forming a thiourea derivative, which is then reacted with an oxidizing agent or a dehydrosulfuration agent, thereby forming a 1,1xe2x80x2dithiophenylene bis(ethylcarbodiimide).
In a particular embodiment, a biscarbodiimide having an intramolecular disulfide bond is represented by Structural Formula (2) or (3): 
Another embodiment is a thiourea derivative having an intramolecular disulfide bond, the thiourea derivative formed by reacting an isothiocyanate with cystamine. In a particular embodiment, a thiourea derivative having an intramolecular disulfide bond is represented by Structural Formula (4): 
Yet another embodiment is a thiourea derivative having an intramolecular disulfide bond, the thiourea derivative formed by reacting an isothiocyanate with 2-aminophenyl disulfide or 4-aminophenyl disulfide, thereby forming the thiourea derivative.
In a particular embodiment, a thiourea derivative having an intramolecular disulfide bond is represented by Structural Formula (5) or (6): 
Yet another embodiment is a urea derivative having an intramolecular disulfide bond, the urea derivative fork by reacting an isocyanate with cystamine. In a particular embodiment, a urea derivative having an intramolecular disulfide bond is represented by Structural Formula (7): 
Another embodiment includes a cross-linked hyaluronan derivative containing at least one intramolecular disulfide bond, wherein the derivative is the product of a reaction between the precursor of the derivative and a biscarbodiimide having an intramolecular disulfide bond. In a particular embodiment, a cross-linked hyaluronan derivative containing at least one intramolecular disulfide bond is the product of a reaction between hyaluronic acid or a salt thereof and a biscarbodiimide having an intramolecular disulfide bond. In another embodiment, a cross-linked hyaluronan derivative containing at least one intramolecular disulfide bond is the product of a reaction between hyaluronic acid or a salt thereof and a biscarbodiimide represented by Structural Formula (1): 
A particular embodiment includes a cross-linked hyaluronan derivative represented by Structural Formula (8) and salts thereof: 
In another embodiment, a thiolated hyaluronan derivative and salts thereof have at least one pendant thiol group, the thiolated hyaluronan derivative formed as a product of a reaction between a cross-linked hyaluronan containing at least one intramolecular disulfide bond and a reducing agent. In a particular embodiment, a thiolated hyaluronan derivative having at least one pendant thiol group may be represented by Structural Formula (9): 
Another embodiment is a compound that may be represented by Structural Formula (10) and salts thereof: 
wherein R is a small molecule or monovalent moiety selected from alkyl, aryl, alkylene, halo, alkyl halide, amine, ethylamine, alkoxy, aryloxy, alkaryloxy, carboxylate, borate, and phenylborate.
Another embodiment is a compound that may be represented by Structural Formula (10) and salts thereof: 
wherein R is a drug or pharmaceutically active moiety.
Another embodiment of the invention is a method of preparing a biscarbodiimide compound represented by Structural Formula (1), including the steps of reacting ethyl isothiocyanate with cystamine, thereby forming a thiourea intermediate, 2,2xe2x80x2dithiobis (N-ethyl(Nxe2x80x2-ethylthiourea)), having Structural Formula (4); and reacting the thiourea intermediate with an oxidizing agent or a dehydrosulfuration agent, thereby forming a biscarbodiimide compound represented by Structural Formula (1).
Another embodiment is a method of preparing a biscarbodiimide compound represented by Structural Formula (1), including the steps of reacting ethyl isocyanate with cystamine, thereby forming a urea intermediate, 2,2xe2x80x2dithiobis (N-ethyl(Nxe2x80x2-ethylurea)), having Structural Formula (7); and reacting the urea intermediate with a dehydrating agent, thereby forming a compound represented by Structural Formula (1).
Another embodiment is a method of preparing a biscarbodiimide compound represented by Structural Formula (2) or (3), including the steps of reacting ethyl isothiocyanate with 2-aminophenyl disulfide or 4-aminophenyl disulfide, thereby forming a thiourea intermediate having Structural Formula (5) or (6); and reacting the thiourea intermediate with an oxidizing agent or a dehydrosulfuration agent, thereby forming 1,1xe2x80x2dithio-o-phenylene bis(ethylcarbodiimide), having Structural Formula (2), or 1,1xe2x80x2dithio-p-phenylene bis(ethylcarbodiimide), having Structural Formula (3).
Another embodiment is a method of preparing a thiolated hyaluronan derivative having Structural Formula (9), comprising the steps of reacting a biscarbodiimide compound represented by Structural Formula (1), with hyaluronic acid or a salt thereof, to form a cross-linked hyaluronic acid derivative of Structural Formula (8); and reacting the derivative of Structural Formula (8) with tris(2-carboxyethyl)phosphine hydrochloride, thereby forming the thiolated hyaluronan derivative having Structural Formula (9).
Yet another embodiment is a method of cross-linking pendant thiol groups on a thiolated hyaluronic acid derivative to form a hydrogel, the method including the step of: reacting a thiolated hyaluronan derivative of structural formula (9), 
with a homobifunctional cross-linker.
The present invention has many advantages. For example, the hyaluronan derivative represented by structural formula (10) and salts thereof, wherein R is a drug or pharmaceutically active moiety is an embodiment which can function as a drug delivery vehicle. The hyaluronan derivative of this embodiment can bind to bioactive agent R without significantly reducing its activity, and is also capable of slowly releasing the bioactive agent at a target tissue site. With such a slow-release delivery vehicle, bioavailability can be more controlled and the dosing kept more even than with many currently available delivery systems. Further, use of a slow-release delivery vehicle allows the amount of drug administered at one time to be kept low to minimize side effects, and the frequency of administration to be reduced.
A hyaluronan derivative according to the invention also provides several advantages when used in ophthalmic surgery and in tissue engineering or tissue regeneration. The hyaluronan derivative according to one embodiment of the invention is a cross-linked, biocompatible, biodegradable material having a sufficiently high viscosity, resilience, other good mechanical properties, and sufficient stability to perform its intended function, but can be decreased in viscosity and decreased in stability. When used in ophthalmic surgery, the derivative can be decreased in viscosity so that it can clear out through the trabecular meshwork and be absorbed by the body. When used as a scaffold to grow tissue, the viscosity of the derivative can be decreased, and as the derivative disintegrates, it can become disassociated from cells grown thereon.